Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Glucocorticoids inhibit transcription of the murine cytoplasmic thymidine kinase gene (Tk-1). Glucocorticoid regulation of Tk-1 transcription can be demonstrated in cells that are arrested in late G1. This observation indicates that inhibition of Tk-1 expression is not dependent upon redistribution within the cell cycle but is due to glucocorticoid regulation of this gene. Transfection studies have been carried out using chimeric genes in which restriction fragments of the Tk-1 promoter were fused to chloramphenicol acetyltransferase or neomycin phosphotransferase. These chimeric reporters were assayed for stable expression and glucocorticoid regulation in P1798 lymphoma cells. A 140-bp fragment, extending from -143 to -3 bp with respect to the thymidine kinase translational start site, was capable of both basal and glucocorticoid-regulated transcription of reporter genes. The extent of inhibition by glucocorticoids was similar to that observed for the endogenous gene, and no increase in basal expression or the extent of inhibition was observed with constructs containing additional 5'-flanking DNA. The 140-bp Tk-1 core promoter fragment binds to transcription factors in extracts from P1798 cells. Control cell extracts contain factors that bind to and protect (from deoxyribonuclease I) a distal promoter element from -106 to -87 bp, relative to the translational start site. A second, proximal element was protected at -43 to -36 bp. The proximal element of the Tk-1 promoter resembles an RNA polymerase II initiator element. No other elements were protected. Glucocorticoids inhibit the amount or activity of the transcription factor that binds to this initiator-like element within the Tk-1 promoter. This element, when fused to upstream activation sequences from the herpes simplex virus thymidine kinase promoter, conveys glucocorticoid sensitivity in cis.
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PMID:Glucocorticoid regulation of a transcription factor that binds an initiator-like element in the murine thymidine kinase (Tk-1) promoter. 896 Dec 64

The herpes simplex virus thymidine kinase gene (HSV-TK) in combination with ganciclovir (GCV), is currently being used in gene therapy-based clinical trials for cancer treatment. Its therapeutic effect is based on a "bystander effect" whereby HSV-TK gene-modified tumor cells are toxic to nearby unmodified tumor cells when exposed to the antiviral drug GCV. We have recently hypothesized that the in vivo mechanism of this bystander effect is due to alterations in the tumor microenvironment in response to release of cytokines and an infiltration of leukocytes after treatment with HSV-TK gene-modified tumor cells and GCV, which results in tumor regression. Expression of B7, a recently identified costimulatory molecule that is important for T-cell stimulation, has been shown to be modulated by stimulatory cytokines interferon-gamma, tumor necrosis factor-alpha, and inhibited by interleukin-10. In the present study, we investigated whether the cytokines released after HSV-TK and GCV treatment could include the expression of the costimulatory molecules B7-1 and B7-2 and the adhesion molecule (ICAM)-1 in the tumor. Furthermore, we investigated whether this altered environment affected the antitumor properties of host lymphocytes. An in vitro model was developed to establish the effects of HSV-TK gene-modified tumor cells and GCV on tumor infiltrating cells. The murine macrophage cell line (IC21) was exposed to either supernatants or cell lysates collected from a mixture of HSV-TK-transduced (KBALB-STK) and non-transduced (KBALB) murine fibrosarcoma tumor cells previously exposed to GCV (experimental). Immunohistochemical analysis showed a significant expression (P < .0001) of B7-1 and B7-2 post exposure of IC21 cells to either supernatant or lysate. In contrast, the level of expression in IC21 cells exposed to the control lysate or supernatant remained unchanged for B7-1 and B7-2. In vivo analysis for B7-1 and B7-2 expression by immunohistochemistry in tumor tissues from experimental mice receiving HSV-TK gene-modified tumor cells and GCV treatment showed a significant expression of B7.1 (35%, P < .0001) and B7.2 (38.2%, P < .0001) on tumor-infiltrating mononuclear cells. In contrast, tumor-bearing control animals showed low levels of B7-2 expression (5.8%), whereas B7-1 was undetectable, as confirmed by reverse-transcriptase polymerase chain reaction. In addition, a significant up-regulation of ICAM expression (50%) on tumor tissues was observed in the experimental group (P = .0317) as compared with the control group (25%). Furthermore, T cells isolated from experimental mice showed a significant in vitro proliferative response (p = .0202) when exposed to syngeneic tumor cells as compared with the control group. These data demonstrated that the use of HSV-TK gene-modified tumor cells and GCV as a suicide gene in the treatment of an intraperitoneal tumor resulted in the expression of the B7 costimulatory molecules and ICAM-1 adhesion molecule and enhanced proliferative response of host T cells. These findings help to understand the mechanism of tumor cell killing in vivo using HSV-TK gene-modified tumor cells.
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PMID:Expression of costimulatory molecules: B7 and ICAM up-regulation after treatment with a suicide gene. 898 40

The expression of herpes simplex virus 1 gamma (late) genes requires functional alpha proteins (gamma1 genes) and the onset of viral DNA synthesis (gamma2 genes). We report that late in infection after the onset of viral DNA synthesis, cell nuclei exhibit defined structures which contain two viral regulatory proteins (infected cell proteins 4 and 22) required for gamma gene expression, RNA polymerase II, a host nucleolar protein (EAP or L22) known to be associated with ribosomes and to bind small RNAs, including the Epstein-Barr virus small nuclear RNAs, and newly synthesized progeny DNA. The formation of these complexes required the onset of viral DNA synthesis. The association of infected cell protein 22, a highly posttranslationally processed protein, with these structures did not occur in cells infected with a viral mutant deleted in the genes U(L)13 and U(S)3, each of which specifies a protein kinase known to phosphorylate the protein.
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PMID:Association of herpes simplex virus regulatory protein ICP22 with transcriptional complexes containing EAP, ICP4, RNA polymerase II, and viral DNA requires posttranslational modification by the U(L)13 proteinkinase. 899 34

Lytic infection of mammalian cells with herpes simplex virus type 1 (HSV-1) results in rapid repression of host gene expression and selective activation of the viral genome. This transformation in gene expression is thought to involve repression of host transcription and diversion of the host RNA polymerase (RNAP II) transcription machinery to the viral genome. However, the extent of virus-induced host transcription repression and the mechanisms responsible for these major shifts in transcription specificities have not been examined. To determine how HSV-1 accomplishes repression of host RNAP II transcription, we assayed transcription patterns on several cellular genes in cells infected with mutant and wild-type HSV-1. Our results suggest that HSV-1 represses RNAP II transcription on most cellular genes. However, each cellular gene we examined responds differently to the transcription repressive effects of virus infection, both quantitatively and with respect to the involvement of viral gene products. Virus-induced shutoff of host RNAP II transcription requires expression of multiple immediate-early genes. In contrast, expression of delayed-early and late genes and viral DNA replication appear to contribute little to repression of host cell RNAP II transcription. Modification of RNAP II to the intermediately phosphorylated (II(I)) form appears unlinked to virus-induced repression of host cell transcription. However, full repression of host transcription is correlated with depletion of the hyperphosphorylated (IIO) form of RNAP II.
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PMID:Repression of host RNA polymerase II transcription by herpes simplex virus type 1. 903 35

Alu interspersed repetitive elements possess internal RNA polymerase III promoters that are transcribed in vitro and in transfected mouse cells but are nearly silent in human HeLa cells. Transcriptional repression of these elements is to some extent reversible, as pol III-dependent Alu expression can be induced with herpes simplex or adenovirus. To assess whether sequence-specific DNA binding proteins might contribute to Alu transcriptional silencing, we examined the internucleosomal spacer region surrounding the B box of the Alu pol III promoter in HeLa cell nuclei for evidence of proteins bound at specific sites in vivo. We identified a DNase I-hypersensitive site 5' to the B box and a DNase I-resistant region 3' to the B box in nuclei. An Alu-specific repressor binds to a 5-bp inverted repeat motif overlapping the 5' end of the TFIIIC binding site and may inhibit pol III transcription through competitive displacement. The level of Alu-specific pol III repressor activity is significantly reduced in adenovirus-infected HeLa cells, suggesting that the repressor may contribute to Alu transcriptional silencing in vivo. The 3' DNase I-resistant region coincided with a binding site for the pol II transcription factor YY1 in vitro. YY1 is one of the major proteins in HeLa cells having binding specificity for Alu elements. YY1 bound to tandem arrays of genomic Alu elements may play a role in chromatin organization and silencing.
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PMID:Specific binding sites for a pol III transcriptional repressor and pol II transcription factor YY1 within the internucleosomal spacer region in primate Alu repetitive elements. 904 Nov 22

Almost all proteins mediating transcriptional activation from promoter-distal sites attach themselves, directly or indirectly, to specific DNA sequence elements. Nevertheless, a single instance of activation by a prokaryotic topologically linked DNA-tracking protein has also been demonstrated. The scope of the latter class of transcriptional activators is broadened in this work. Heterologous fusion proteins linking the transcriptional activation domain of herpes simplex virus VP16 protein to the sliding clamp protein beta of the Escherichia coli DNA polymerase III holoenzyme are shown to function as topologically DNA-linked activators of yeast and Drosophila RNA polymerase II. The beta:VP16 fusion proteins must be loaded onto DNA by the clamp-loading E. coli gamma complex to be transcriptionally active, but they do not occupy fixed sites on the DNA. The DNA-loading sites of these activators have all the properties of enhancers: they can be inverted and their locations relative to the transcriptional start site are freely adjustable.
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PMID:Activation of RNA polymerase II by topologically linked DNA-tracking proteins. 919 31

Lymphocytic myocarditis is thought to be a virus-induced disease. T cells expressing the alpha-beta T-cell receptor seem to play a central role in the pathogenesis and to mediate tissue injury in this disease. A case of active fulminant myocarditis is described, which was analyzed by immunohistochemical, molecular biologic, and serologic methods. Infiltration of the heart tissue predominantly by gamma-delta T cells was detected by immunohistochemistry. No evidence of viral disease could be obtained by in situ hybridization with different enterovirus-specific DNA probes; by reverse-transcriptase polymerase chain reaction using specific primers for enteroviruses, adenoviruses, herpes simplex viruses, influenza A and B viruses, and cytomegaloviruses; or by enzyme-linked immunosorbent assay and electron microscopy. Because gamma-delta T cells may have an autoimmune capacity, we propose that these cells may trigger autoimmune myocarditis. These findings may be important in order to identify subgroups of patients who may benefit from immunosuppressive therapy.
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PMID:Active fulminant myocarditis characterized by T-lymphocytes expressing the gamma-delta T-cell receptor: a new disease entity? 929 89

Nuclear distribution and migration of herpes simplex virus type 1 Us11 transcripts were studied in transient expression at the ultrastructural level and compared to that of RNA polymerase II protein. Transcription was monitored by autoradiography following a short pulse with tritiated uridine. Us11 transcripts accumulated mainly over the foci of intermingled RNP fibrils as demonstrated by the presence of silver grains localizing incorporated radioactive uridine superimposed to these structures in which the presence of Us11 RNA and poly(A) tails was previously demonstrated. Silver grains were also scattered over the remaining nucleoplasm but not in the clusters of interchromatin granules, and over the dense fibrillar component of the nucleolus as in control, nontransfected HeLa cells. Pulse-chase experiments revealed the transient presence of migrating RNA in the clusters of interchromatin granules. RNA polymerase II was revealed by immunogold labeling following the use of two monoclonal antibodies: mAb H5, which recognizes the hyperphosphorylated form of the carboxy-terminal domain (CTD) of the molecule, and mAb 7C2, which recognizes both its hyperphosphorylated and unphosphorylated forms. The two mAbs bind to the newly formed Us11 transcription factories and the clusters of interchromatin granules of transfected cells. In control cells, however, clusters of interchromatin granules were labeled with mAb H5 but not with mAB 7C2. Taken together, our data demonstrate the involvement of the clusters of interchromatin granules in the intranuclear migration of Us11 RNA in transient expression. They also suggest the occurrence of changes in the accessibility of the RNA polymerase II CTD upon expression of the Us11 gene after transfection by exposing some epitopes, otherwise masked in nontransfected cells.
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PMID:Identification of transcription factories in nuclei of HeLa cells transiently expressing the Us11 gene of herpes simplex virus type 1. 936 2

Previously, we described a nonviral cytoplasmic gene therapy vector system based on the T7 autogene concept. This system has been shown to achieve rapid and high levels of gene expression in a variety of animal cells and tissues. To test the utility of the system in vivo tumor ablation, a T7 cancer gene therapy plasmid vector, pT7T7/T7TK, was constructed. This nonviral vector contains a T7 autogene, T7T7, and a human herpes simplex virus thymidine kinase (HSV-TK) gene driven by a second T7 promoter (T7TK). When co-transfected with T7 RNA polymerase (T7 RNAP) into cultured human osteosarcoma 143B cells, abut 10-20% of the cells were found to express HSV-TK, and more than 90% of the cells were killed in the presence of 1 microM ganciclovir (GCV) within 4 days after DNA transfection. The increase in killing above the transfection frequency is due to a "bystander" effect among transfected and untransfected 143B cells. Direct injections of pT7T7/T7TK into 143B tumors grown in nude mice resulted in TK gene expression in tumor cells located near the injection sites as revealed by the immunohistochemical staining. Repeated tumor injections of the pT7T7/T7TK vector and intraperitoneal (i.p.) injections of GCV resulted in inhibition of tumor growth and in tumor shrinkage in 6 out of 10 treated nude mice. Three of those six tumors fully regressed shortly after the end of the GCV injections. All of the full tumor regressions were found to be permanent and no apparent tumor relapses were observed for the rest of the lives of the treated nude mice after the initial tumor ablations. These results, combined with the nonviral and rapid cytoplasmic gene expression features, suggest that the T7 vector may be a good candidate for cancer gene therapy and other medical and biological applications.
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PMID:Cancer gene therapy by direct tumor injections of a nonviral T7 vector encoding a thymidine kinase gene. 955 20

Transcriptional activators can stimulate multiple steps in the transcription process. We have used GAL4 fusion proteins to characterize the ability of different transcriptional activation domains to stimulate transcriptional elongation on the hsp70 gene in vitro. Stimulation of elongation apparently occurs via a mechanistic pathway different from that of stimulation of initiation: the herpes simplex virus VP16, heat shock factor 1 (HSF1) and amphipathic helix (AH) activation domains all stimulate initiation, but only VP16 and HSF1 stimulate elongation; and mutations in hydrophobic residues of the HSF1 activation domains impair stimulation of elongation but not of initiation, while mutations in adjacent acidic residues impair stimulation of initiation more than of elongation. Experiments in which activators were exchanged between initiation and elongation demonstrate that the elongation function of HSF1 will stimulate RNA polymerase that has initiated and is transcriptionally engaged. Transcriptional activators thus appear to have at least two distinct functions that reside in the same domain, and that act at different times to stimulate initiation and elongation.
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PMID:Transcriptional activation domains stimulate initiation and elongation at different times and via different residues. 960 96


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